Abstract
Brucellosis is an emerging infection in marine mammals in Brazil, where serological and molecular evidence of the infection has been reported in members of Delphinidae and Pontoporiidae.1 On 11 September 2019, an adult (246 cm of body length) cachectic male of Kogia sima was found dead-stranded (moderate decomposition, COD 3) on Corumbau-Prado, Bahia State, Northeast Brazil (16°53’36.636”S, 39°6’58.896”W), during the cetacean stranding monitoring conducted by Projeto Baleia Jubarte. During necropsy, selected tissue samples were collected for brucellosis diagnosis: heart, lung, spleen, and rete mirabile were fixed in 10% formalin for histopathology and immunohistochemistry (IHC), using a polyclonal B. abortus antibody (Byorbyt Ltd®, 1:3000); brain, testicle, thyroid, kidney, spinal cord, spleen, prescapular and pulmonary lymph node, lung, liver, urinary bladder, and heart were frozen at -20°C for microbiological and molecular tests.
Of frozen tissues, 1:3 (w/v) suspensions prepared with sterile 0.9% NaCl solution had the DNA extracted and PCR-amplified targeting Brucella insertion sequence (IS711).2 Tissue suspensions were plated on Farrell’s and CITA agar3,4 and incubated at 37°C under 10% CO2 for 4 weeks. Colonies morphologically compatible with Brucella5 had the DNA extracted and tested by PCR directed to bcsp31 and bp26 genes for the identification of marine Brucella species (B. ceti and B. pinnipedialis)6,7. Brucella DNA positive samples were further PCR-amplified and sequenced with primers directed to aroA gene.8
Gross findings included: multifocal epidermal abrasions; purulent secretion on the left auditive channel; pulmonary and tracheal edema and hemorrhage; multifocal ulcers on keratinized stomach; and intense parasitosis on all gastric compartments and blubber. The mandible and a portion of skin and muscular tissue from the flank were removed with parallel cuts (cutting object). Microscopically, diffuse pulmonary edema and multifocal splenic hemosiderosis were noted. IHC was negative.
In brain and spinal cord Brucella DNA was detected by IS711-PCR and Brucella suspected colonies were recovered in CITA medium. Bacterial colonies yielded positive results in bcsp31-PCR and a 1900 bp amplicon in bp26-PCR indicating a marine strain. The partial sequence of aroA gene assigned the isolate as Brucella. Although the histopathological analysis of the central nervous system was not available, the bacterial isolation suggests neurobrucellosis, a condition that can be directly associated with stranding and death.
To our knowledge, this report comprises the first isolation of Brucella in cetaceans in the South Atlantic Ocean. In Kogiidae, anti-Brucella antibodies were reported in two K. breviceps specimens in Japan.9 B. ceti zoonotic genotype ST27 was isolated in a pregnant K. sima, stranded at the pacific coast of Costa Rica in 2018.10 A complete phenotypic and genotypic characterization of the Brucella isolate obtained in Brazil will be performed.
This report reinforces the relevance of systematic monitoring of zoonotic pathogens in cetaceans to address the relevance of the infections for species conservation and to protect human health. Handling of marine mammals should be performed by professional responders using appropriate protection equipment and local population must be aware of the risks of manipulating those animals.
Acknowledgements
Projeto Baleia Jubarte is sponsored by Petróleo Brasileiro S.A. (Petrobras). This study was financed in part by the Coordination for the Improvement of Higher Education Personnel—Brazil (CAPES)—Finance Code 001. A.M.S.S. is the recipient of a postdoctoral fellowship (PNPD/CAPES). S.C.S. is the recipient of a PhD fellowship (CAPES). L.B.K, R.M.S, and J.L.C.D. are the recipients of a fellowship by the National Research Council (CNPq; grants #312036/2018-3, #310532/2017-5, and #304999/2018-0, respectively).
*Presenting author
+Student presenter
Literature Cited
1. Sánchez-Sarmiento AM, Carvalho VL, Díaz-Delgado J, Ressio RA, Fernandes NCCA, Guerra JM, Sacristán C, Groch KR, Silvestre-Perez N, Ferreira-Machado E, Costa-Silva S, Navas-Suárez P, Meirelles ACO, Favero C, Marigo J, Bertozzi CP, Colosio AC, Marcondes MCC, Cremer MJ, Silva N dos S, Neto JSF, Keid LB, Soares R, Sierra E, Fernández A, Catão-Dias JL. 2019. Molecular, serological, pathological, immunohistochemical and microbiological investigation of Brucella spp. in marine mammals of Brazil reveals new cetacean hosts. Transbound Emerg Dis 66(4):1674–1692.
2. Batinga MCA, De Lima JTR, Gregori F, Diniz JA, Muner K, Oliveira TMFS, Ferreira HL, Soares RM, Keid LB. 2018. Comparative application of IS711-based polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) for canine brucellosis diagnosis. Mol Cellular Probes 39:1–6.
3. OIE World Organization for Animal Health. 2012. Bovine Brucellosis. Man Diagnostic Tests Vaccines Terr Anim. Paris (France): OIE. 1–35.
4. De Miguel MJ, Marín CM, Muñoz PM, Dieste L, Grilló MJ and Blasco JM. 2011. Development of a selective culture medium for primary isolation of the main Brucella species. J Clin Microbiol 49(4):1458–1463.
5. Alton CG, Jones IM, Angus RD, Verger JM. 1998. Techniques for the brucellosis laboratory. Paris: Institut National de la Recherche Agronomique. 190.
6. Baily GG, Krahn JB, Drasar BS, Stoker NG. 1992. Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hygiene 95:271–275.
7. Cloeckaert A, Grayon M, Grepinet O. 2000. An IS711 Element downstream of the bp26 gene is a specific marker of Brucella spp. isolated from marine mammals. Clin Diag Lab Immunol 7(5):835–839.
8. Whatmore AM, Perrett LL, MacMillan AP. 2007. Characterization of the genetic diversity of Brucella by multilocus sequencing. BMC Microbiol 34:4–15.
9. Ohishi K, Katsumata E, Uchida K, Maruyama T. 2007. Two stranded pygmy sperm whales (Kogia breviceps) with anti-Brucella antibodies in Japan. Vet Rec 160(18):628–629.
10. Suárez-Esquivel M, Ruiz-Villalobos N, Hernández-Mora G, González-Barrientos R, Palacios-Alfaro JD, Barquero-Calvo E, et al. 2019. Brucella sequence Type 27 isolated from Dwarf Sperm Whale (Kogia sima) stranded in the Costa Rican Pacific Coast. Microbiol Soc 1:1A.